DESCRIPTION: Metalloproteins have many different functions in cells, such as storage and transport of small molecular substrates, proteins, enzymes and signal transduction proteins. In these metalloproteins, the interplay between the metal oxidation state/coordination geometry and the overall protein conformation plays important roles in their functions. The long term objective of the proposed research is to gain new insight into correlations between metal active site structures of a metalloenzyme and their functions through high resolution simultaneous structural snapshots during different biological relevant processes, such as electron transfer, ligand binding and protein folding. The proposed research aims at building instrumentation for mapping reaction trajectories on multiple spatial and temporal scales. Transient electronic and nuclear structures of metal centered active sites in a series of metalloproteins will be simultaneously captured using X-ray transient absorption (XTA, or transient X-ray absorption spectroscopy) spectroscopy with time resolutions from 10-13 second (100 fs) to longer, while the protein conformation change along the reaction coordinates can be captured by X-ray transient scattering (XTS). The method uses a laser pulse pump to trigger a biological reaction, which can be direct photodissociation of ligands/inhibitors or photoinduced redox reaction at metal active sites that subsequently triggers protein conformation changes, and an X-ray pulse to probe the active site structures and the conformations of proteins as a function of the delay time between the laser and X-ray pulses. The proposed research focuses on building a portable and multifunctional sample chamber/detection setup to enable simultaneous detection of the local/long range structures of metalloproteins during enzymatic reactions. The proposed setup will be built upon an existing X-ray facility with advanced detectors and laser systems to fulfill specific needs for protein samples. Once the instrumentation is built, it will be a part of portabl instrumentation for conducting experiments in other light sources, such as the linear coherence light source (LCLS) with femtosecond pulsed X-rays, and a part of shared instrumentation with other users. Three metalloprotein systems will be investigated using the instrumentation built through the proposed work: 1) metal binding site structures and protein conformations of cytochrome c during folding/refolding; 2) conformation gating in electron transfer of hybrid hemoglobins; and 3) transient active site structures in catalase. These structural results combined with those of reaction kinetics from other transient spectroscopic measurements will provide much deeper understanding of energy transduction inside the proteins during enzymatic reactions and guidance for modulating protein functions via structural modifications around the active sites, which will lead to advances in enzymatic function enhancement, catalysis, as well as theoretical calculations.